miR-107靶向细胞周期蛋白E1对人非小细胞肺癌A549细胞功能的影响研究
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摘要
目的探讨miR-107对非小细胞肺癌A549细胞功能的影响及可能的靶基因。方法实验分为脂质体+miR-107模拟物过表达组(OV-miR-107组)、脂质体+miR-107抑制模拟物下调组(KD-miR-107组)和脂质体+阴性对照模拟物组(NC组),siRNA的细胞转染分别为siRNA-1、siRNA-2、siRNA-3。MTT法检测细胞增殖能力,流式细胞实验检测细胞周期,双荧光素酶报告基因实验检测miR-107下游靶基因,实时定量逆转录PCR和Western blot分别检测下游靶基因mRNA和蛋白表达。结果miR-107呈时间和剂量依赖性地抑制A549细胞的增殖(P<0.05);miR-107阻滞更多A549细胞停留在G0G1期,而S期和G2M期占比下降(P<0.05);miR-107结合于细胞周期蛋白E1(CCNE1)的3′-UTR区域246~253bp位点,并降低CCNE1mRNA和蛋白表达(P<0.05);A549细胞中下调CCNE1后,siRNA-2抑制细胞增殖(P<0.05)并阻滞细胞停留在G0G1期(P<0.05)。结论 miR-107靶向调节CCNE1抑制人非小细胞肺癌A549细胞增殖和调控细胞周期。
Objective To investigate the effect of miR-107 on the function of human non-small cell lung cancer cell line A549 and its possible target genes.Methods The experiment was divided into the liposome+miR-107 mimics overexpression group(OVmiR-107 group),liposome+miR-107 inhibiting mimics downregulation group(KD-miR-107 group)and liposome+negative control mimics group(NC group).The cell transfections of siRNA were siRNA-1,siRNA-2 and siRNA-3 respectively.The cellular proliferation capacity was detected by MTT assay.The cellular cycle was detected by flow cytometry.The dual luciferase reporter gene assay was performed to detect the downstream target gene of miR-107.The real time quantitative reverse transcription PCR and Western blot were respectively employed to examine mRNA and protein expression levels of downstream target gene.Results miR-107 inhibited the proliferation of A549 cells in a time-and dose-dependent manner(P<0.05).miR-107 arrested more A549 cells at the G_0G_1 phase,and the proportions of S phase and G2M phase were decreased(P<0.05).miR-107 combinded with the 246 bp-253 bp of CCNE1 3′-untranslated region,and decreased mRNA and protein expression of CCNE1(P<0.05);after down-regulating CCNE1 in A549 cells,siRNA-2 inhibited cellular proliferation(P<0.05)and blocked the cells to stay at G_0G_1 phase(P<0.05).Conclusion miR-107 inhibits the proliferation of human non-small cell lung cancer cell line A549 and regulates the cellular cycle by targeting CCNE1.
Objective To investigate the effect of miR-107 on the function of human non-small cell lung cancer cell line A549 and its possible target genes.Methods The experiment was divided into the liposome+miR-107 mimics overexpression group(OVmiR-107 group),liposome+miR-107 inhibiting mimics downregulation group(KD-miR-107 group)and liposome+negative control mimics group(NC group).The cell transfections of siRNA were siRNA-1,siRNA-2 and siRNA-3 respectively.The cellular proliferation capacity was detected by MTT assay.The cellular cycle was detected by flow cytometry.The dual luciferase reporter gene assay was performed to detect the downstream target gene of miR-107.The real time quantitative reverse transcription PCR and Western blot were respectively employed to examine mRNA and protein expression levels of downstream target gene.Results miR-107 inhibited the proliferation of A549 cells in a time-and dose-dependent manner(P<0.05).miR-107 arrested more A549 cells at the G_0G_1 phase,and the proportions of S phase and G2M phase were decreased(P<0.05).miR-107 combinded with the 246 bp-253 bp of CCNE1 3′-untranslated region,and decreased mRNA and protein expression of CCNE1(P<0.05);after down-regulating CCNE1 in A549 cells,siRNA-2 inhibited cellular proliferation(P<0.05)and blocked the cells to stay at G_0G_1 phase(P<0.05).Conclusion miR-107 inhibits the proliferation of human non-small cell lung cancer cell line A549 and regulates the cellular cycle by targeting CCNE1.
引文
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[2]SIEGEL R L,MILLER K D,JEMAL A.Cancer statistics,2016[J].CA Cancer J Clin,2016,66(1):7-30.
[3]SCHABATH B,NGUYEN A,WILSON P,et al.Temporal trends from 1986to 2008in overall survival of small cell lung cancer patients[J].Lung Cancer,2014,86(1):14-21.
[4]BARTEL D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[5]MARCO G,JORGE C B,MARTA C,et al.Exosomal miRNA analysis in non-small cell lung cancer(NSCLC)patients′plasma through qPCR:A feasible liquid biopsy Tool[J].J Vis Exp,2016(111):e53900.
[6]HU Z,CHEN J,TIAN T,et al.Genetic variants of miRNA sequences and non-small cell lung cancer survival[J].J Clin Invest,2008,118(7):2600-2608.
[7]LIN Y,YANG C.Circulating miRNA signature for early diagnosis of lung cancer[J].EMBO Mol Med,2011,3(8):436-437.
[8]CHEN L,LI Y,XU Y,et al.Upregulation of miR-107inhibits glioma angiogenesis and VEGF expression[J].Cell Mol Neurobiol,2016,36(1):113-120.
[9]ZHANG L,MA P,SUN M,et al.MiR-107down-regulates SIAH1expression in human breast cancer cells and silencing of miR-107inhibits tumor growth in a nude mouse model of triple-negative breast cancer[J].Mol Carcinog,2016,55(5):768-777.
[10]ZHANG M,WANG X,LI W,et al.miR-107and miR-25simultaneously target LATS2and regulate proliferation and invasion of gastric adenocarcinoma(GAC)cells[J].Biochem Biophys Res Commun,2015,460(3):806-812.
[11]XIA H,LI Y,LV X H.MicroRNA-107inhibits tumor growth and metastasis by targeting the BDNF-mediated PI3K/AKT pathway in human non-small lung cancer[J].Int J Oncol,2016,49(4):1325-1333.
[12]ZHANG Z,ZHANG L,YIN Y,et al.miR-107regulates cisplatin chemosensitivity of A549non small cell lung cancer cell line by targeting cyclin dependent kinase 8[J].Int J Clin Exp Pathol,2014,7(10):7236-7241.
[13]HAN Z Y,ZHANG Y B,YANG Q Y,et al.miR-497and miR-34aretard lung cancer growth by co-inhibiting cyclin E1(CCNE1)[J].Oncotarget,2015,6(15):13149-13163.
[14]KOUTSAMI M K,TSANTOULIS P K,KOULOUKOUSSA M,et al.Centrosome abnormalities are frequently observed in non-small-cell lung cancer and are associated with aneuploidy and cyelin Eoverexpression[J].J Pathol,2006,209(4):512-521.